NMDA receptors play a central role in synaptic plasticity and neuropathology, and are known targets of ethanol. Two signal transduction systems that regulate plasticity and survival are the ERK and PKB cascades, and we have recently shown that NMDA receptors control ERK and PKB activity through a common signaling module. Furthermore, this control is bidirectional, and involves activation of opposing stimulatory and inhibitory pathways. While the components of the stimulatory pathway have been substantially defined, the nature of the inhibitory pathway remains obscure. An important down-stream effector of ERK/PKB signaling is CREB, and it was recently reported that NMDA receptors show a similar bidirectional control of CREB activity. Importantly, synaptic NMDA receptors promote survival signaling through CREB, whereas extrasynaptic NMDA receptors activate a CREB shut-off pathway and promote neuronal death. We now hypothesize that the ERK/PKB activation pathway is coupled to synaptic NMDA receptors, while the inhibitory pathway is coupled to extrasynaptic NMDA receptors. Furthermore, preliminary data is presented showing that chronic ethanol induces marked changes in the distribution of NMDA receptors between synaptic and extrasynaptic sites. It is proposed that these changes will have profound effects on ERK, PKB, and CREB (EPC) signaling by altering the balance between the stimulatory and inhibitory pathways. The over-arching goal of this proposal is to determine the effects of ethanol on NMDA receptor trafficking between synaptic and non-synaptic sites, and upon regulation of EPC signaling by NMDA receptors. This project will utilize a well-defined neuronal cell culture model and employ various experimental techniques including whole-cell patch-clamp electrophysiology, biochemical and molecular assays, confocal microscopy, and fluorescence immunohistochemistry. The Specific Aims are to: (1) Determine the effects of acute ethanol on the modulation of CREB activity by synaptic and extrasynaptic NMDA receptors; (2) Test the hypothesis that inhibition of CREB by extrasynaptic NMDA receptors involves upstream inhibition of ERK/PKB; (3) Test the hypothesis that chronic ethanol induces a redistribution of NMDA receptors from extrasynaptic to synaptic sites and leads to corresponding changes in EPC signaling; and (4) Test the hypothesis that ethanol-induced changes in NMDA receptor localization and EPC signaling lead to changes in neuronal plasticity and survival. Our preliminary data showing chronic ethanol-induced change in the trafficking of NMDA receptors together with compartmentalization of discrete signaling complexes have important implications for understanding how ethanol leads to enduring changes in the brain.